Screening and characterization of endophytic
Bacillus
and
Paenibacillus
strains
from medicinal plant
Lonicera japonica
for use as potential plant growth promoters
Longfei Zhao
1,*, Yajun Xu
1, Xin-He Lai
2,*, Changjuan Shan
3,
Zhenshan Deng
4, Yuliang Ji
51
Key Laboratory of Plant-Microbe Interactions of Henan, Shangqiu Normal University, PR China. 2
Institute of Translational Medicine, Wenzhou Medical University, PR China. 3
School of Science and Technology, Henan Institute of Science and Technology PR China. 4
College of Life Sciences, Yan’an University, PR China. 5
Biological and Medical Engineering Department, Shangluo University, PR China.
Submitted: January 13, 2014; Approved: December 28, 2014.
Abstract
A total of 48 endophytic bacteria were isolated from surface-sterilized tissues of the medicinal plant
Lonicera japonica, which is grown in eastern China; six strains were selected for further study based on their potential ability to promote plant growth in vitro (siderophore and indoleacetic acid produc-tion). The bacteria were characterized by phylogenetically analyzing their 16S rRNA gene similarity, by examining their effect on the mycelial development of pathogenic fungi, by testing their potential plant growth-promoting characteristics, and by measuring wheat growth parameters after inocula-tion. Results showed that the number of endophytic bacteria inL. japonicavaried among different tis-sues, but it remained relatively stable in the same tissues from four different plantation locations. Among the three endophytic strains, strains 122 and 124 both had high siderophore production, with the latter showing the highest phosphate solubilization activity (45.6 mg/L) and aminocyclo-propane-1-carboxylic acid deaminase activity (47.3 nmol/mg/h). Strain 170 had the highest indo-leacetic acid (IAA) production (49.2 mg/L) and cellulase and pectinase activities. After inoculation, most of the six selected isolates showed a strong capacity to promote wheat growth. Compared with the controls, the increase in the shoot length, root length, fresh weight, dry weight, and chlorophyll content was most remarkable in wheat seedlings inoculated with strain 130. The positive correlation between enzyme (cellulose and pectinase) activity and inhibition rate onFusarium oxysporum, the IAA production, and the root length of wheat seedlings inoculated with each tested endophytic strain was significant in regression analysis. Deformity of pathogenic fungal mycelia was observed under a microscope after the interaction with the endophytic isolates. Such deformity may be directly related to the production of hydrolytic bacterial enzymes (cellulose and pectinase). The six endophytic bac-terial strains were identified to be Paenibacillus andBacillus strains based on the results of 16S rRNA gene sequencing analysis and their physiological and biochemical characteristics. Results in-dicate the promising application of endophytic bacteria to the biological control of pathogenic fungi and the improvement of wheat crop growth.
Key words: Lonicera japonica, Bacillus, Paenibacillus, plant growth-promoting characteristics, endophytic bacterium, wheat (Triticum aestivum).
DOI: http://dx.doi.org/10.1590/S1517-838246420140024
Send correspondence to L. Zhao. Shangqiu Normal University, Shangqiu, 476000 Henan, PR China. E-mail: hnzhaolongfei@163.com. *These authors contributed equally to this work.
Introduction
Endophytes are important constituents of the plant microecosystem. These organisms have formed a mutually beneficial relationship with their host plants during long-term evolutionary processes. Endophytic bacteria reside intercellularly or intracellularly within the host tissues and do not cause visible damage or morphological changes in their hosts. Therefore, these bacteria may be more advanta-geous for plant survival as protection from environmental stress and microbial competition (Geetha et al., 2008). Some endophytes benefit the host plants,i.e., they increase soil porosity, produce indoleacetic acid (IAA, a phyto-hormone), siderophores, and antibiotic compounds, func-tion in phosphate solubilizafunc-tion and nitrogen fixafunc-tion, suppress phytopathogens via competition for colonization sites and nutrients, and act as antagonists of nematodes (Khan et al., 2008). Furthermore, endophytes may help symbiotic rhizobia form nodules with non-specific hosts and promote plant growth (Zhaoet al., 2011). Therefore, endophytic bacteria are important microbial resources and have gradually become a multidisciplinary research hotspot in different fields, including botany, microbiology, plant protection, and plant breeding. Microorganisms associated with medicinal plants are of interest as producers of com-pounds responsible for plant bioactivity. A recent study has highlighted the potential use of endophytes for synthesis of bioactive compounds, promotion of plant growth, and en-hanced resistance to various pathogens and drought (Miller
et al., 2012a). Conceivably, endophyte-derived metabolites may be related to the observed bioactivity and beneficial health claims of the host plants in traditional Chinese medi-cine (TCM). As a perennial semi-evergreen winding woody liana of the genus Lonicera in Caprifoliaceae,
Lonicerae japonica(commonly known as honeysuckle or
Jinyinhua in Chinese) is an herb used in TCM as Flos Lonicerae; the plant is widely grown in the Henan, Hebei, and Shandong Provinces of China. Given its latent fe-ver-clearing, antibacterial, antifungal, antiviral, and anti-inflammatory effects, the herb has been prescribed to treat “fever syndrome” (a TCM term; an aspect of the common cold), febrile diseases, dysentery, carbuncles, and virulent swellings (Mphprc 2000; Liet al., 2003; Wuet al., 2007).
Bacteria has been isolated from the rhizosphere of wheat (Triticum aestivum), which is a major staple crop be-ing cultivated worldwide and the largest annual crop (24.4 Mha, 2012) in China. These bacterial isolates mainly in-clude Azospirillum brasilense, Pantoea agglomerans,
Arthrobacter spp., Achromobacter xylosoxidans,
Herbaspirillum hiltneri, Stenotrophomonas maltophilia,
Pseudomonas spp., Bacillus spp., Rahnella aquatilis,
Paenibacillus riograndensis, and P. polymyxa(Venieraki
et al., 2011).BacillusandPaenibacillusstrains are promi-nent members of the endophyte population in healthy tis-sues of medicinal plants and function as prolific producers of bioactive compounds, including antimicrobials,
sidero-phores, and phytotoxins (Milleret al., 2012b). For exam-ple,P. polymyxabenefits plants by reducing disease sever-ity, inducing defense mechanisms, promoting growth, and producing several hydrolytic enzymes (b-1,3-glucanases and chitinases) and antifungal or antibacterial metabolites (Denget al., 2011; Razaet al., 2009; Laiet al., 2012; Raza
et al., 2009). In addition, Bacillus sp. is involved in the biosynthesis of a broad spectrum of antibiotics (e.g., sur-factant lichenysin) and can effectively reduce disease inci-dence in diverse plant hosts by inducing systemic disease resistance (Biancoet al., 2011), forming biofilms on root surfaces and endospores, suppressing root phytopathogens, and promoting heat and desiccation tolerance by colonizing the rhizosphere (Chenet al., 2013; Liuet al., 2009).
Phytopathogens cause a variety of plant diseases and are useful model microorganisms for studying various as-pects of host-pathogen interactions. Alternaria and
Fusariumspecies can be found as pre-harvest fungal con-taminants in wheat (Maríaet al., 2013).Alternariasp. is the predominant genus found in wheat grown in different agroecological regions, withA. alternatabeing the most prevalent species (Gonzalez et al., 1996; Broggi et al., 2007; Ramirez et al., 2005; María et al., 2013).
Magnaporthe griseacauses rice blast disease, which is one of the most serious plant fungal diseases, whereas
Fusarium oxysporummay cause diseases in various crops, such as rice and wheat.
The endophytes present in various plants and the anti-bacterial activity of endophytic fungi isolated from Flos Loniceraehave been investigated (Li et al., 2010). How-ever, only a few studies have reported the effect of endophytic bacteria from Flos Lonicerae on phyto-pathogens (Xuet al., 2013), and information is limited on the use of endophytes isolated from tissues of the medicinal plantL. japonicain wheat production. Moreover, the role of endophytic bacteria in plant growth and their antagonis-tic potential against phytopathogens are unclear.
The objectives of our study were as follows: (1) to isolate and screen endophytic bacteria from tissues of the medicinal plantL. japonica; (2) to characterize the plant growth-promoting characteristics (PGPC) of endophytic bacteria; and (3) to detect the antifungal activities and ef-fects of endophytic bacteria on wheat seedlings.
Materials and Methods
Collection ofL. japonicasamples
County of Henan Province, and the Pingyi County and Juye County of Shangdong Province. The distance between sampling sites was more than 50 km; each sampling site in-cluded at least three subsites that were more than 1 km apart. From each subsite, 5 plants that were separated at least by 30 m were randomly chosen and uprooted. Upon collection, samples were placed in sterile bags and stored in the dark at 4 °C until further processing, usually within 24 h of collection.
Isolation of endophytic bacteria
Briefly, 5 g each of root, stem, or leaf were carefully weighed from a pooled mixture of healthy L. japonica
plants, washed with sterile water to remove remaining soil particles and attached epiphytic bacteria, and cut into 1-2 cm small portions with sterile scissors. These portions were further surface-sterilized by sequential immersion in 95% ethanol for 30 s then in 5% sodium hypochlorite for 3 min before the samples were finally rinsed eight times in sterile distilled water. A total of 5 plants were used from each subsite to form a mixture sample from the same tissue, such that 15 plants were used for each site. Strain isolation was performed from a pooled replicate of the same tissue from five plants in each subsite, such that each site had three replicates. The surface-sterilized portions were placed into sterile metal mortar, ground to slurry with 0.85% sterile saline, and shaken with a vortex for 1 min. In the stationary state, the supernatant was collected and di-luted in different concentrations of the bacterial suspen-sion. Subsequently, 100mL of each processed sample sus-pension was plated (with three replicates per sample) on nutrient agar (NA) plates (5.0 g peptone, 1.5 g yeast extract, 1.5 g beef extract, 5.0 g NaCl, 20 g agar, and 1 L distilled water; pH 7.2) (Denget al., 2011). The cultures were incu-bated at 28 °C for 3 d. A single colony of each isolate was re-streaked on fresh plates of the same media and micro-scopically examined. The pure cultures were preserved on plates at 4 °C for temporary storage or in sterile vials with 30% (v/v) glycerol for long-term storage at -80 °C.
To confirm the successful surface sterilization pro-cess, the surface sterilized portions were rolled over the NA plates or aliquots of water from the final rinse solutions. These portions were plated onto fresh NA plates and exam-ined for contaminants. Plates with no contaminants were effectively surface sterilized and were used for the isolation of endophytes.
Characterization of plant growth-promoting characteristics (PGPC) of endophytic bacteria
Examination of siderophore production
Bacteria were cultured in lysogeny broth (LB; 10 g NaCl/L) for 72 h under iron-restricted conditions. Aliquots of each bacterial culture were inoculated in plates (three plates per strain) containing agar Chrome Azurol S (CAS)
and incubated at 30 °C. Plates were observed daily for 7 d to detect the appearance of an orange halo around the colonies (Schwynet al., 1987). The siderophore levels produced by the isolates corresponded to the diameter of the orange halo. The presence of catechol and hydroxamate siderophores in the culture supernatants obtained from bac-teria grown in iron-restricted conditions in casamino acid (CAA) medium was quantitatively determined by the colo-rimetric assay, as previously described (Maet al., 2011).
Phosphate solubilization
To determine the phosphate-solubilizing activity, the isolates were cultured in triplicate in modified Pikovskayas medium (0.5% tricalcium phosphate) (Sundara-Raoet al., 1963) at 30 °C for 7 d at 200 rpm. The solubilized phos-phate in the culture supernatant was quantified as described by Fiske and Subbarow (1925).
Indole acetic acid (IAA) production
IAA production was examined as previously de-scribed (Gordonet al., 1951; Inéset al., 2011). Briefly, each bacterial suspension (1 x 108cfu/mL) was inoculated in 10 mL LB broth containing L-TRYPTOPHAN (100mG/ML) AND INCUBATED AT 28 °C for 72 h at 200 rpm. Bacterial cells were removed by centrifugation at 8,000 rpm for 15 min, and the collected supernatant was in-cubated at room temperature in the dark for 30 min. Pure IAA (Sigma, USA) was used as a standard. The IAA con-centration in the culture supernatant was calculated accord-ing to the optical density of the culture, which was measured at 530 nm with Salkowski’s reagent (12 g/L FeCl3in 7.9 M H2SO4). Each experiment was performed thrice.
Aminocyclopropane-1-carboxylic acid (ACC) deaminase activity
0.1 M Tris-HCl buffer (pH 8.5) was incubated for 30 min at 30 °C before adding with 1 mL of 0.56 M HCl. The mixture was centrifuged at 12000 rpm for 5 min to obtain the supernatant. Subsequently, 400 mL of 0.56 M HCl and 150 mL of 0.2% 2,4-dinitrophenylhydrazine in 2 M HCl were added to 500mL of the supernatant. The mixture was incubated for 30 min at 30 °C before adding 1 mL of 2 M NaOH. The amount ofa-ketobutyrate was measured by de-termining the optical density at 600 nm with mixtures with-out the cell suspension or ACC as the controls.
Cellulase and pectinase activity
The activities of cellulase and pectinase were assayed on indicator plates. For the cellulase assay, nitrogen-freebase (NFB) plates supplemented with 0.2% carboxy-methyl cellulose and 0.5% tryptone were spotted with bac-terial cells. After incubation for 48 h at 30 °C, the plates were coated with a Congo Red (1 mg/mL) solution for 30 min. The excess stain was discarded, and the agar was destained with 1 M of a NaCl solution. Plates were kept overnight at 4 °C and examined on the following day for clearing zones around the points of inoculation.
For the pectinase assay, the bacterial isolates were spotted on NA medium supplemented with 0.5% pectin, and the plates were incubated at 30 °C. On the fifth day of incubation, a 2% cetyltrimethyl ammonium bromide (CTAB) solution was added to the plate surface for 30 min and then discarded. The plates were washed with 1 M NaCl to visualize the zone around the bacterial growth (Maet al., 2011).
In vitro detection of antifungal activity
The interaction of endophytes with pathogenic fungi (F. oxysporum,M. grisea, andA. alternata) was performed via the point inoculation method as previously described (Zhaoet al., 2011). Briefly, a small block of agar with fun-gal growth was cut from potato dextrose agar (PDA) plates (extract of 200 g potato with 20 g glucose, 18 g agar, and 1 L distilled water) by a sterile puncher (Ø = 4 mm). Each block was placed in the center of a fresh PDA plate. Test strains were spot-inoculated on the edge of PDA plate (ap-proximately 25 mm from the center), incubated at 28±2 °C for 7 d, and observed for zones of inhibition, with fungal mycelia cultivated for 7 d without spot inoculation as con-trol (Geethaet al., 2008). Experiments were performed in triplicate for each bacterial isolate.
Microscopy of fungal mycelia
The morphological changes caused by endophytes on the mycelia of each pathogenic fungal species after cultur-ing for 4 d on PDA plates were directly examined, photo-graphed under an optical fluorescence microscope (BX50 Olympus; under 200x magnification), and compared with the structures of the control groups.
Effects of endophyte inoculation on plant growth
Wheat seeds (Triticum aestivumcv. ‘Zhoumai 18’, a national authorized wheat variety of China) were surface sterilized with 100% alcohol for 1 min, 5% sodium hypo-chlorite for 3 min, and finally rinsed six times with sterile distilled water. Surface-sterilized seeds were allowed to germinate axenically in Petri dishes filled with moist filter paper at 28 °C. The surface-sterilized seeds were immersed for 3 h in a thick suspension of the exponential phase bacte-ria (approximately 109-1010cfu/mL) cultured in 5 mL of YM broth. The germinated seeds were grown in pots filled with sterilized vermiculite, which was moistened with ster-ile water, as described by Vincent (1970). Seeds were cul-tured under greenhouse conditions programmed for a 14 h/d photoperiod at a constant temperature of 28 °C dur-ing the day and 20 °C at night at approximately 50% rela-tive humidity. All pot experiments were incubated with isolates 122, 124, 130, 132, 135, or 170 in three repetitions with 30 seedlings per pot. Seedlings without bacteria were used as the controls. The wheat plants were harvested after 7 weeks when seedling roots were well developed. Parame-ters such as the fresh weight, dry weight, shoot length, root length, and the chlorophyll content of experimental plants were measured and compared with those of the control plants (plants that were not inoculated with endophyte).
Estimation of chlorophyll content
The greenest leaves were collected from 20-day-old wheat plants without the dry leaves, plant diseases, and in-sect pests and were then kept in the dark. A total of 90 plants were used in the replicates, and 3 repetitions were performed. From each plant, three leaves were collected, cut into smaller pieces, and mixed. Subsequently, 1 g of leaf tissue was crushed in a mixture of ethanol and acetone (v/v, 1:1) and maintained at 40 °C for 24 h. The chlorophyll con-tent was spectrophotometrically measured with the specific absorption coefficients for chlorophyll a and b at 645 and 663 nm, respectively. The chlorophyll content was calcu-lated according to Geethaet al.(2008), with the mixture of ethanol and acetone as the control.
Identification of endophytic bacterial strains
Strain morphology, physiological characteristics, and biochemical tests
Sequencing and phylogenetic analysis
The phylogeny of the 16S rRNA genes has been used as one of the main criteria for differentiating species, gen-era, and higher taxa in current bacterial taxonomy. To iden-tify the potential endophytic bacteria, total genomic DNA was extracted from the isolates as previously described (Moulinet al., 2004). The 16S rRNA gene was selectively amplified from genomic DNA by PCR with the universal forward primer P1 (5’-CGGGATCCAGAGTTTGATCC TGGCTCAGAACGAACGCT-3’) and reverse primer P6 (5’-CGGGATCCTACGGCTACCTTGTTACGACTTCA CCCC-3’), which corresponded to the positions 8-37 bp and 1479-1506 bp, respectively, in theEscherichia coli16S rRNA gene (van Berkumet al., 1996). An aliquot of PCR products of isolates was directly sequenced by the Sangon Biotech (Shanghai) Co., Ltd. with the same primers men-tioned above. The acquired and related sequences were matched with the ClustalX 1.81 software and manually cor-rected with Bioedit 4.8.4. A phylogenetic tree was constructed with the Jukes-Cantor model and the neigh-bor-joining method (Saitouet al., 1987) by the TREECON software package (van de Peeret al., 1997). The computa-tion of the similarity of each strain tested was performed by the DNAMAN application (version 6.0.3.40; Lynnon Cor-poration). The obtained 16S rRNA gene sequences were
deposited in the NCBI GenBank
(http://www.ncbi.nlm.nih.gov/) under the accession num-bers KC208613 through KC208618.
Statistical analysis
Data on the density of endophytic bacteria in different tissues ofLonicera japonica, growth promotion, and endo-phytic inoculation experiments were treated with ANOVA (analysis of variance), and mean comparisons were per-formed with Tukey’s test (p = 0.05). Regression analysis was performed with the IBM SPSS 17.0 package (by the Data Theory Scaling System Group, Faculty of Social and Behavioral Sciences, Leiden University, The Netherlands).
Results
Isolation of endophytic bacteria fromL. japonica
Endophytic bacteria (48 strains) with different colony morphology (e.g., shape, size, and color) were isolated from the healthy root, stem, and leaf tissues ofL. japonica
plants. By contrast, no colonies appeared on the NA plates upon incubation at 28 °C for 2 d to 3 d, either by rolling over with the sterilized surface portions of this medicinal plant or plating aliquots of water from final rinse solutions, thereby indicating successful surface sterilization. The amount of endophytic bacteria significantly varied between the different tissues, as follows: 3.62 x 104cfu/g in roots, 0.88 x 104cfu/g in stems, and 2.73 x 104cfu/g in leaves (Ta-ble 1). No significant differences were observed in the bac-terial density between the same tissues among different plants from all four plantation locations (p = 0.05).
Characterization of endophytic bacteria for factors with plant growth-promoting potential
All 48 strains were studied for their siderophore pro-duction. Six of these strains showed remarkable perfor-mance as evidenced by the orange halo around the colony. The color change is attributed to iron removal by the bacte-ria from the blue CAS-Fe (III) complex in the CAS agar medium with the dark blue background. Only 5 of the 6 strains except strain 132 produced a siderophore concentra-tion in the range of 1.8-87.2 mg/L for catecholate and 0.90-76.3 mg/L for hydroxymate (Table 2) in the quantita-tive analysis. Strains 122 and 124 showed the most signifi-cant siderophore production after 24 h of incubation.
The phosphate solubilization potential was studied for all 48 isolates, only 6 isolates showed a zone of phos-phate solubilization on the Pikovskayas agar medium with tricalcium phosphate. These 6 strains were further quanti-fied for phosphate solubilization in a liquid medium; 4 of which were within the range of 1.87-45.6 mg/L (Table 2). Isolated strain 124 had the highest level of phosphate solu-bilization, whereas isolates 135 and 170 showed levels that were under the detection limit.
IAA production was measured for all the 48 endo-phytic bacteria in the range of 11.5-49.2 mg/L after 72 h of
Table 1- Density of endophytic bacteria isolated from different tissues ofL. japonica(x 104cfu/g).
Origin sites Roots Stems Leaves
Shangqiu, Henan Province 3.56±0.0038a, § 0.87±0.0048a 2.98±0.0051a
Huaxian, Henan Province 3.89±0.0038b 0.96±0.0089b 2.61±0.0029b
Pingyi, Shangdong Province 3.67±0.0092c 0.83±0.0060c 2.87±0.0062c
Juye, Shangdong Province 3.37±0.0142d 0.86±0.0021a 2.45±0.0092d
Mean 3.62±0.0078 0.88±0.0055 2.73±0.0059
incubation (Table 2). The IAA levels of the strains in the culture supernatant matched their anti-fungal activity. Iso-late 170 produced significantly higher IAA levels than the other isolates.
Among the tested 48 isolates, only 4 strains could uti-lize ACC as the sole carbon source (Table 2), which is an indication of the ACC deaminase activity. Among these 4 strains, strains 124 and 122 were the top two strains that uti-lize ACC as the sole carbon source. Based on the develop-ment of a yellow-color zone on the NFB and NA plates, all six strains exhibited cellulase and pectinase activities, with isolate 170 showing the highest levels (Table 2). The statis-tical differences between strains were shown in Table 2 in terms of the activities of ACC deaminase, cellulase, and pectinase, as well as phosphate solubilization and IAA/siderophore production.
Detection ofin vitroantifungal activity
The inhibitory activity of the 48 endophytic bacterial strains against pathogenic fungi (M. grisea,F. oxysporum, andA. alternata) was measured. All six selected strains (12.5%) strongly inhibited pathogenic fungi compared with the control. Based on the inhibition of mycelial growth, strain 124 showed the strongest effect againstF. oxysporum
(74.39%), followed by strain 132 against A. alternata
(70.93%), and strain 130 againstM. grisea(73.75%), re-spectively (Table 3). Compared with the controls, the cor-relation was positive and significant between the activities of hydrolytic enzymes (cellulose and pectinase) and the in-hibition rate ofF. oxysporum(Figure 1A-C) for each strain tested.
Microscopy of pathogenic fungi mycelia
Compared with the nearly straight and even mycelia of the control under laboratory condition and cultured on PDA medium for 4 d (Figure 2A-C; also shown in the Sup-plementary figure), the colonies of the pathogenic fungi
shrunk upon interaction with endophytic bacteria, and their mycelia underwent considerable morphological changes, as follows: becoming coralline (Figure 2Da), fractured (Figure 2Fd, f), swollen, globular, or atrophied (Figure 2Fd-f); forming a bending knot (Figure 2Eb-c); and under-going autolysis (Figure 2Fe).
Wheat growth promotion after endophyte inoculation
The six bacterial isolates with plant growth-pro-moting potential and antagonistic activity against patho-genic fungiin vitrowere further evaluated because of their effectiveness in promoting wheat growth uponin vivo inoc-ulation. Compared with the control, strain 130 significantly increased its shoot length by 14.57% (Figure 3A), whereas all six strains promoted root length to a different extent. Root length promotion was more pronounced with strains 130, 132, and 170 (10.87%, 8.42%, and 10.27%, respec-tively as shown in Figure 3B). Strains 130 and 135
signifi-Table 2- PGPC of endophytic bacteria.
Strains Sid. (C) (mg/L)*
Sid. (H) (mg/L)
Pho. (mg/L)
IAA production (mg/L)
ACC deaminase (nmol/mg/h)
Cellulase activity (D/d)
Pectinase activity (D/d)
122 61.6±0.17a§ 76.3±0.56a 35.3±0.26a 18.3±0.12a 45.2±0.36a 1.20±0.08a 1.34±0.01a
124 87.2±0.36b 56.1±0.26b 45.6±0.46b 11.5±0.12b 47.3±0.26b 1.14±0.05a 1.20±0.06b
130 41.7±0.53c 45.5±0.53c 14.9±0.72c 29.3±0.35c 35.6±031c 0.56±0.03c 0.59±0.05c
132 - - 1.87±0.07d 22.9±0.14d - 0.76±0.03d 0.82±0.01d
135 1.8±0.06d 0.90±0.09d - 12.6±0.17e 12.3±0.39d 1.36±0.03b 1.43±0.02a
170 21.9±0.36e 23.4±0.26e - 49.2±0.46f - 2.58±0.06e 2.64±0.04e
Control / / / - / -
-Note: Sid. (C), Siderophore production (Catechol-type); Sid. (H), Siderophore production (Hydroxamate-type); Pho phosphate solubilization; IAA, indoleacetic acid; ACC, 1-aminocyclopropane-1-carboxylic acid, activity (nmola-ketobutyrate/mg biomass/h. D/d indicates the ability to produce siderophores, cellulose, and pectinase. D, diameter of colony and halo; d - colony diameter.
*
Average (±, standard deviation), the data in columns is average values of three repetitions. §
The same letter means no significant differences between treatments at 0.01 level; negative action, blank; control for IAA assay was LB (10 g NaCl/L) without inoculated bacterial suspension under the same incubation condition.
Table 3- Inhibition of endophytic bacteria by pathogenic fungi.
Strains Source F. oxysporum A. alternata M. grisea
122 Root 2.9 64.63* 4.6 46.51* 3.4 57.50*
124 Root 2.1 74.39 5.6 34.89 2.3 71.25
130 Stem 6.6 19.51 3.3 61.63 2.1 73.75
132 Leaf 6.5 20.73 2.5 70.93 4.2 47.50
135 Root 3.3 59.76 3.1 63.95 4.1 48.75
170 Stem 2.8 65.85 3.1 63.95 2.9 63.75
Control / 8.2 0 8.6 0 8.0 0
Notes: Data are the mean of three samples. Colony diameter, cm.
*Inhibition ratio of pathogenic fungus (%) = (Control colony diame-ter-treatment colony diameter)100/ Control colony diameter.
cantly increased wheat fresh weight (16.48% and 15.78%, Figure 3C) and dry weight (20.07% and 19.65%, Figu-re 3D). RegFigu-ression analysis (FiguFigu-re 1A) showed a signifi-cant positive correlation between IAA production and
increase in root length of the wheat seedlings inoculated with endophytic bacteria.
Compared with the uninoculated control (Figure 4), the chlorophyll content of the endopyhte-inoculated wheat
increased to a range of 8.4% to 33.98%, with the strain 130-treated group showing the highest content (33.98%). The inoculation of strain 130 induced the highest increase in shoot length, root length, fresh weight, dry weight, and chlorophyll content of wheat.
Identification of the six plant growth-promoting endophytic bacteria
Physiological and biochemical tests were conducted, including measurements of catalase, V-P test, utilization of carbohydrate, the production of enzymes, growth tempera-ture, salt tolerance, and bacterial morphology (i.e., size, shape, and Gram staining; Supplementary Table 1). Based on these results and the sequencing of 16S rRNA gene and phylogeny analysis (Figure 5 and Table 4), our endophytic isolates belonged to two genera, namely, Bacillus and
Paenibacillus. Strains 122 and 130 showed high identity with Paenibacillus and were most closely related to P. polymyxa IAM 13419T (D16276) and P. ehimensis
KCTC3748 (AY116665) with 98.7% and 100% similarity, respectively. Therefore, a Paenibacillus sub-clade was formed. Strains 170, 124, 132, and 135 had high sequence similarities toB. atrophaeusNRRLNRS-213T(EU138516)
(99.6%), B. megaterium IAM13418T (D16273) (99.1%), andB. subtilisFL (EU221673) with a similarity of 99.6% and 99.7%.
Discussion
This work is the first report on the isolation and popu-lation density of endophytic bacteria from the medicinal plantL. japonica, which is widely planted in the Henan and Shandong provinces in eastern China. As summarized in Table 1, the endophytic bacterial load varied significantly in different tissues, but it remained relatively stable without significant differences in the same tissues of different plants from different planting locations (p = 0.05), thereby implying that the prevalence of endophytic bacteria de-pended on the plant tissues being colonized and the micro-environment they lived in. The endophytic bacterial number in roots is the highest probably because the root soil environment is fairly complex under the effect of both bi-otic and abibi-otic factors. Previous studies have reported that the population of endophyticB. subtilisE1R-j in roots in-creases when the bacterial cell number reisolated from the leaf tissue distinctly decreases from the second to fourth
Figure 2- Morphological changes of the mycelia of plant pathogenic fungi upon interaction withLonicera japonicaendophytes. Images in A, B, and C were representative of normal mycelia ofM. griseaMG01,F. oxysporumFO02, andA. alternataAA03. Images in D, E, F were the atrophy and deformity ofM. griseaMG01 mycelia (by strain 130), bending knot ofF. oxysporumFO02 mycelia (by strain 124), autolysis, fracture, and atrophy ofA. alternata
leaf stage, thereby indicating thatB. subtilisE1R-j can relo-cate from the root to the aerial parts of a wheat plant (Liuet al., 2009). Our results are in line with those obtained in a re-cent report (Gaoet al., 2012) that endophytic bacterial
den-sity varies in different parts of the plant, with the highest value in roots and the lowest value in stems. Similar pat-terns of bacterial population levels in the root and stem tis-sues have been widely reported in cabbage (B. subtilisBB),
Figure 3- Effect of six endophytic bacteria on shoot length (A), root length (B), fresh weight (C), and dry weight (D) of wheat seedlings. Each value is the mean of ten replicates. Bars represent the standard deviations of mean. Statistical significance was determined at p < 0.05 according to the Tukey’s test. The asterisk represents significant differences.
cacao (B. subtilis), rose, and crops such as maize, wheat, rice (B. subtilisstrain NR-64), soybean, sweet corn, sugar beet, and potato, or in various medicinal plants such as
Glycyrrhizaspp.,Pinellia ternate,Lycium chinense, Digi-talis purpurae, Leonurus heterophyllus, Bletilla striata,
Belamcanda chinases, P. pedatisecta, and Taxus
yunnanensis (Venieraki et al., 2011; Gao et al., 2012; Wulffet al., 2003; Bahiget al., 2012; Liet al., 2012; Miller
et al., 2012a; Leiteet al., 2013).
Siderophore production could confer competitive ad-vantages in bacteria for colonizing plant tissues, excluding other microorganisms from the same ecological niche
Figure 5- Neighbor-joining tree based on the alignment of nucleotide sequences of the 16S rRNA gene from the tested strains (shown in bold) and refer-ence strains. GenBank accession numbers were placed in parentheses. Bootstrap values greater than 50% were indicated. Scale bar represents the number of substitutions per site.
Table 4- Identification and classification of the tested strains.
Strains Genus affiliation Accession No. of the 16S rDNA sequence Best closest match Similarity (%)
122 Paenibacillus KC208613 Paenibacillus polymyxaIAM 13419T(D16276) 98.7
124 Bacillus KC208614 Bacillus megateriumIAM13418T(D16273) 99.1
130 Paenibacillus KC208617 Paenibacillus ehimensisIFO15659T(AB021184) 100
132 Bacillus KC208615 Bacillus subtilisFL (EU221673) 99.6
135 Bacillus KC208616 Bacillus subtilisFL (EU221673) 99.7
(Loaceset al., 2011), competing for nutrients, and protect-ing plant from phytopathogens (Compant et al., 2005). Metagenomic analysis (Sessitschet al., 2012) revealed that the presence of a high number of genes involved in sidero-phore production in an endophyte community that colo-nizes rice roots indicates a strong biocontrol capacity because endophytes compete with other pathogens for iron. Among the 6 endophytic bacteria (3 strains from roots, 2 strains from stems, and 1 strain from leaves; 4Bacillusand 2 Paenibacillus strains), strains 122 and 124 showed a higher capacity for siderophore production.
Several phosphate-solubilizing microorganisms are able to convert insoluble phosphorus to a soluble form through acidification, secretion of organic acids or protons (Richardsonet al., 2009), or chelation and exchange reac-tions (Hameedaet al., 2008; Bhattacharyyaet al., 2012), thereby representing a possible mechanism of direct plant growth promotion under field conditions (Verma et al., 2001). These microorganisms are important for plant nutri-tion because they increase phosphate uptake and act as biofertilization promoters of wheat crops.Bacillus is re-portedly one of the most significant phosphate-solubilizing bacteria (Mehnazet al., 2006). Four of our six strains are capable of phosphate solubilization. Strains 124 (Bacillus) and 122 (Paenibacillus) showed relatively higher phos-phate solubilization than the others, and these strains signif-icantly increased the dry weight and fresh weight of the inoculated wheat, respectively.
In this report, six endophytic strains from the medici-nal plantL. japonicaexhibited inhibitory activity (Table 3) against phytopathogenic fungi (F. oxysporum,M. grisea,A. alternata), which are useful model organisms for studying various aspects of host-pathogen interactions. These patho-gens are chosen as test targets in the antifungal activity ex-periment because of their capacity to cause epiphytic disease and major damage in crops and plants, includingL. japonicaand wheat. Previous works have verified that pro-moting plant growth and inhibiting phytopathogen growth may involve a large number of bacterial endophytes. A re-cent report also suggests that the cellulase and pectinase produced byKlebsiella oxytocaGR-3 might play an impor-tant role in plant-microbe interactions and the intercellular colonization of roots (Maet al., 2011). Our findings re-vealed that all 6 of the selected strains (Bacillus and
Paenibacillus) exhibited cellulose and pectinase activities. Growth-promoting agents can enhance plant growth and have the potential to replace the use of chemical fertil-izers, pesticides, and other supplements (Kimet al., 2011; Bhattacharyyaet al., 2012). Endophytes that are associated with medicinal plants are of interest as producers of com-pounds responsible for the observed plant bioactivity with biosynthetic potential (Milleret al., 2012b). This finding is in agreement with our result,i.e., strain 130 induced the largest increase in root length, stem length, flesh weight, dry weight, and the chlorophyll content of wheatin vivo,
al-though itsin vitroACC deaminase activity is lower than that of strains 122 and 124. One explanation for this phe-nomenon is that strain 130 contains ACC deaminase but its activity was not induced very much in thein vitrostudy. Al-ternatively, strain 130 showed a higher IAA production, thereby directly promoting wheat growth via hormonal stimulation. Furthermore, plant growth is the overall result of all growth-promoting molecules produced by root endo-phytes and ectoendo-phytes (Pattenet al., 2002).
In summary, our work showed that some of the endo-phytic bacteria (strains 130, 135, and 170) isolated from the medicinal plant L. japonica can produce wheat growth-promoting moleculesin vitro and increase wheat growth (i.e., root length, stem length, flesh weight, dry weight, and chlorophyll content)in vivo. Therefore, these endophytic bacteria have the potential to be used as plant growth-promoting agents in agriculture to increase crop growth.
Acknowledgments
This work was supported by projects from the Na-tional Science Foundation of China (U1204301), the Henan Provincial Education Department of Science and Technol-ogy Research Key Research Project (12A210019), and the Foundation for University Key Teacher by the Ministry of Education of Henan Province (2012GGJS166). The Au-thors are grateful to Dr. Guihong Yin for providing the wheat seeds (cv. `Zhoumai18’). We thank Prof. Yi Ren of the Department of Biomedical Sciences, Florida State Uni-versity College of Medicine, Tallahassee, FL, USA, Dr. Jane Kelly, of the Medical Research Service, RD-33, De-partment of Veterans Affairs Medical Center, 3710 SW U.S. Veterans Hospital Road, Portland, OR, USA, and an anonymous reviewer from the USA for proofreading and polishing the final draft of our manuscript.
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Supplementary Material
Figure S1. Antagonistic activity of endophytes against phyto-pathogenic fungi after 3 d. (A) Inhibition ofM. griseaMG01 by endophyte strain 130; (B0 inhibition ofF. oxysporum FO02 by endophyte strain 124; C. inhibition ofA. alternata AA03 by endophyte strain 132. A’, B’, and C’ were control pathogenic fungusfungal colonies without endophytes. Table S1. A Physiological and biochemical test results and cell
characteristics of strains 122, 124, 130, 132, 135, and 170.
Associate Editor: Lara Durães Sette
